Evidence for an ester linkage between the labile binding site of C3b and receptive surfaces.
Evidence for an ester linkage between the labile binding site of C3b and receptive surfaces.
复制标题
C3b 不稳定结合位点与接受表面之间存在酯键的证据。
作者：
S. Law;N. Lichtenberg;R. Levine
C3 b的不稳定结合位点通过共价键与受体表面相互作用。我们在这里报告的实验旨在阐明的联系的性质。125 I-C3 b与表面带有C3-转化酶活性的酵母聚糖（Z）结合。用1%SDS洗涤所得酵母聚糖-C3 b颗粒（Z-C3 b），使其不含非共价结合的蛋白质。将Z-C3 b颗粒在不同pH下孵育，发现C3 b可以在碱性条件下释放，但不能在酸性条件下释放。在1 M羟胺存在下，在碱性pH下也可观察到C3 b的释放。在pH 7.5的1 M羟胺中和在pH 10的0.1 M NaHCO 3中研究了从Z释放C3 b的动力学。在羟胺中，中性pH下C3 b从Z中释放的二级速率常数为0.0026 ± 0.003 M-1 min-1，在pH 10下为37 ± 3 M-1 min-1。这些值与含酯化合物如苯甲酰甘氨酸乙酯、乙酰胆碱和N，O-二乙酰丝氨酰胺的羟氨解和水解的速率常数相当。然而，它们与含有硫酯、酪氨酰酯和酰基咪唑的化合物的羟氨解和水解得到的值显著不同。这些实验的结果与C3 b和感受表面之间的键是酯的假设是一致的。通过分析羟胺从酵母聚糖释放C3 d的产物，获得了C3 b和受体表面之间的酯键的进一步证据。如果键确实是酯，则用羟胺从Z释放C3 d将产生与C3 d或与Z缔合的异羟肟酸盐。我们能够检测到0.66 ± 0.12摩尔异羟肟酸/摩尔C3 d肽，这表明C3 b和感受表面（RS）之间的键呈RS-O-CO-C3 b的形式。
The labile binding site of C3b interacts with receptive surfaces by way of a covalent bond. We report here experiments directed toward elucidating the nature of the linkage. 125I-C3b was bound to zymosan (Z) carrying C3-convertase activity on its surface. The resulting zymosan-C3b particles (Z-C3b) were washed free of noncovalently bound proteins with 1% SDS. The Z-C3b particles were incubated at different pH's, and it was found that C3b can be released under alkaline conditions but not acidic conditions. Release of C3b can also be observed at alkaline pH in the presence of 1 M hydroxylamine. The kinetics of the release of C3b from Z were studied at pH 7.5 in 1 M hydroxylamine and at pH 10 in 0.1 M NaHCO3. The second order rate constants for the release of C3b from Z were found to be 0.0026 ± 0.003 M-1 min-1 in hydroxylamine at neutral pH, and 37 ± 3 M-1 min-1 at pH 10. These values are comparable to the rate constants for the hydroxylaminolysis and hydrolysis of compounds containing esters such as benzoyl glycine ethyl ester, acetylcholine, and N,O-diacetylserinamide. They are significantly different, however, from the values obtained for the hydroxylaminolysis and hydrolysis of compounds containing thioesters, tyrosyl esters, and acylimidazole. The results of these experiments are consistent with the hypothesis that the bond between C3b and receptive surfaces is an ester. Further evidence for an ester linkage between C3b and receptive surfaces was obtained by analyzing the products of the release of C3d from zymosan by hydroxylamine. If the bond is indeed an ester, the release of C3d from Z with hydroxylamine will yield an hydroxamate that either associates with C3d or with Z. We were able to detect 0.66 ± 0.12 moles of hydroxamate per mole of C3d peptide, suggesting that the bond between C3b and receptive surfaces (RS) takes the form of RS-O-CO-C3b.